N1,n12-diacetylspermine as tumor marker

ABSTRACT

A tumor marker comprising diacetylspermine, and a method of evaluating the state of a tumor, comprising reacting an antibody to diacetylspermine with a biological sample to thereby detect diacetylspermine and evaluating the state of the tumor using the obtained detection results as an indicator.

TECHNICAL FIELD

The present invention relates to a tumor marker comprisingN¹,N¹²-diacetylspermine, a method of evaluating the state of a tumorusing the tumor marker, an antibody to diacetylspermine, a method ofdetecting a tumor using the antibody, and a kit for detecting a tumor.

BACKGROUND ART

Polyamine is a general term for those alkylamines with two or more aminogroups. There are four types of polyamines [putrescine (H₂N(CH₂)₄NH₂),cadaverine (H₂N(CH₂)₅NH₂), spermidine (H₂N(CH₂)₄NH(CH₂)₃NH₂) andspermine (H₂N(CH₂)₃NH(CH₂)₄NH(CH₂)₃NH₂)] and acetylated forms thereof inthe human body.

Relatively recently, it was found that two types of diacetylpolyamines[N¹,N⁸-diacetylspermidine (hereinafter expressed as “DiAcSpd”) andN¹,N¹²-diacetylspermine (hereinafter expressed as “DiAcSpm”)] areexcreted in urine though very small in quantities. While thesecomponents occupy only 1.4% and 0.6% of the total polyamine,respectively, in the urine of healthy persons, the ratios of thesecomponents remarkably increase in the urine of cancer patients ascompared to other polyamine components. Further, it has been shown thatthese components have other characteristics of tumor markers (Sugimoto,M. et al., J. Cancer Res. Clin. Oncol., 121, 317-319 (1995); Hiramatsu,K. et al., J. Cancer Res. Clin. Oncol., 123, 539-545 (1997)).

Initially, DiAcSpd and DiAcSpm were quantitatively determined by amethod which was a combination of a fractionation system by HPLC and adetection system using enzyme (Hiramatsu, K. et al., J. Biochem., 117,107-112 (1995)). However, more simple measuring methods have beendeveloped. In particular, with respect to the measurement of DiAcSpm, anELISA method using a specific antibody was developed recently(Hiramatsu, K. et al., J. Biochem., 124, 231-236 (1998)). However,preparation of a kit containing an ELISA measuring system has not beenachieved yet.

Techniques for measuring DiAcSpm by immunoassay have also been developed(Japanese Patent Unexamined Publication No. H11-75839; Japanese PatentUnexamined Publication No. 2000-74917), but they have room forimprovement in terms of measurement sensitivity and cross-reactivity.

It is well known that metabolism of polyamines are activated inassociation with cell proliferation. In fact, polyamine contents tend toincrease in various cancer tissues as compared to normal tissues. Sinceany of these polyamines is contained abundantly in activelyproliferating tissues and is excreted in large quantity in the urine ofcancer patients as compared to the urine of healthy persons, they areevaluated as tumor markers. A kit which is capable of measuring thetotal polyamine content easily by an enzymatic determination methodwithout discriminating acetylated polyamines and free polyamines inurine and without discriminating the four types of polyamines in urinehas already been commercialized (Labo-Search PolyamineAuto; AITCorporation). This kit is used as one of clinical test methods. However,with respect to urinary total polyamine, it has become clear that arelatively large number of false negative results are found in malignanttumor patients and that total polyamine significantly increases inassociation with not only malignant tumors but also with variousdiseases and conditions such as inflammatory diseases, cardiacinfarction, cirrhosis, process of curing of wounds, etc. Thus, it isconsidered that total polyamine cannot be evaluated as a marker specificto malignant tumors (Shunichiro Kubota, NIPPON-RINSHO (Japan ClinicalMedicine) 53, Special Issue, pp. 501-505 (1995)). On the other hand,researchers are now revealing that some of the problems total polyaminehas can be avoided by measuring diacetylpolyamines (DiAcSpd and DiAcSpm)discretely (Sugimoto, M. et al., J. Cancer Res. Clin. Oncol., 121,317-319 (1995)).

DISCLOSURE OF THE INVENTION

The present invention aims at providing DiAcSpm as a tumor marker. Thepresent invention also aims at providing an antibody which is extremelysmall in cross-reactivity with DiAcSpm analogues and yet bindsspecifically to a trace amount of DiAcSpm.

As a result of intensive and extensive researches toward the solution ofthe above-described problems, the present inventors have found thatDiAcSpm is useful as a tumor marker and succeeded in creating anantibody which does not cross-react with other urinary polyamines (thatare DiAcSpm analogues) and reacts only with DiAcSpm specifically. Thus,the present invention has been achieved.

The present invention relates to the following.

-   (1) A tumor marker comprising N¹,N ¹²-diacetylspermine.-   (2) A method of detecting a tumor, comprising reacting an antibody    to N¹,N¹²-diacetylspermine with a biological sample.

In the above method, urine may be used as the biological sample. As thetumor, at least one selected from the group consisting of urinary tractmalignant tumors, colorectal cancer, breast cancer, pancreatic cancer,biliary tract cancer, lung cancer, liver cancer, uterine cervix cancer,brain tumor, myelogenous leukemia and malignant lymphoma may be given.The state of a tumor is at least one selected from the group consistingof the presence/absence of cancer, the degree of progression of cancer,the degree of malignancy of cancer, the presence/absence of metastasisof cancer and the presence/absence of recurrence of cancer.

In the present invention, it is possible to evaluate early cancers(e.g., stages 0 to I in colorectal cancer and stages I to II in breastcancer).

-   (3) An antibody to DiAcSpm, which has at least one property selected    from the following (a) and (b):

(a) cross-reactivity with N¹-AcSpd: 0.1% or less

(b) total cross-reactivity with DiAcSpm analogues present in urine: 5%or less.

As the above antibody, a polyclonal antibody or monoclonal antibody maybe given.

-   (4) A method of detecting DiAcSpm, comprising reacting the above    antibody with a biological sample (e.g., urine).-   (5) A method of detecting a tumor, comprising reacting the above    antibody with a biological sample (e.g., urine). As the tumor, at    least one selected from the group consisting of urogenital malignant    tumors, colorectal cancer, breast cancer, pancreatic cancer, biliary    tract cancer, lung cancer, liver cancer, uterine cervix cancer,    brain tumor, myelogenous leukemia and malignant lymphoma may be    given. In the present invention, it is possible to evaluate early    cancers (e.g., stages 0 to I in colorectal cancer and stages I to II    in breast cancer).-   (6) A tumor detection kit comprising an antibody to    N¹,N¹²-diacetylspermine.

As the antibody used in the kit of the present invention, an antibodyhaving at least one property selected from the following (a) and (b) maybe given:

(a) cross-reactivity with N¹-AcSpd: 0.1% or less

(b) total interference on the measurement results caused by itscross-reaction with DiAcSpm analogues present in urine: 5% or less.

The antibody may be a polyclonal antibody or monoclonal antibody.

As the tumor to be detected, at least one selected from the groupconsisting of urogenital malignant tumors, colorectal cancer, breastcancer, pancreatic cancer, biliary tract cancer, lung cancer, livercancer, uterine cervix cancer, brain tumor, myelogenous leukemia andmalignant lymphoma may be given.

Further, the kit of the present invention has at least one propertyselected from the following (a) to (c):

(a) lower detection limit in actual measurement: 9.06 nM

(b)within-run reproducibility: CV=10% or less

(c) between-day reproducibility: CV=10% or less.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows graphs showing the amounts of polyamines excreted in urine(ratios to cut-off values).

FIG. 2 is a diagram showing polyamines and their mono- and di-acetylatedforms.

FIG. 3 is a diagram showing steps of gradual affinity purification.

FIG. 4 is a diagram showing the cross-reactivity of anti-DiAcSpmantibody.

FIG. 5 is a diagram showing the results of dilution test.

FIG. 6 is a diagram showing the results of dilution test.

FIG. 7 is a diagram showing the results of dilution test.

FIG. 8 is a diagram showing correlation with the values obtained byHPLC.

FIG. 9 is a diagram showing the results of detection of colorectalcancer and breast cancer.

FIG. 10 is a diagram showing the cut-off values of urinary DiAcSpm.

FIG. 11 is a diagram showing the relation between pancreatic/biliarytract diseases and the amount of urinary DiAcSpm.

FIG. 12 is a diagram showing the positive ratios of tumor markers inbenign and malignant diseases.

FIG. 13 is a diagram showing the positive ratios of tumor markersagainst recurrence of malignant tumors.

FIG. 14 is a diagram showing the relation between stages ofpancreatic/biliary tract cancers and urinary or serum tumor markers.

FIG. 15 is a diagram showing the relation between stages ofpancreatic/biliary tract cancers and urinary DiAcSpm concentrations.

FIG. 16 is a diagram showing the time course of urinary DiAcSpm levelswhich reflect the conditions and treatment effects of a patient withprimary malignant lymphoma o the brain.

FIG. 17 is a diagram showing the time course of urinary DiAcSpm levelswhich reflect the conditions and treatment effects of a patient withprimary malignant lymphoma of the brain.

FIG. 18 is a diagram showing the time course of urinary DiAcSpm levelswhich reflect the conditions and treatment effects of an astrocytomapatient.

BEST MODES FOR CARRYING OUT THE INVENTION

Hereinbelow, the present invention will be described more specifically.

The present invention is characterized by using DiAcSpm as a tumormarker. The present invention also relates to an antibody that does notreact with urinary polyamines other than DiAcSpm (DiAcSpm analogues suchas N¹-AcSpd, N⁸-AcSpd, N¹,N⁸-DiAcSpd and AcSpm: see Table 1) and reactsonly with DiAcSpm specifically.

The antibody of the invention has one or both of the followingproperties: (i) its cross-reactivity with N¹-AcSpd (which is present inurine as a DiAcSpm analogue in an amount about 30-times greater thanDiAcSpm) is 0.1% or less; (ii) total interference on the measurementresults caused by its cross-reaction with DiAcSpm analogues present inurine is 5% or less. The numerical value mentioned in (i) above is avalue showing the nature of the antibody. The numerical value mentionedin (ii) above is a value obtained by multiplying cross-reactivity itselfby the abundance of these analogues in urine; this value can be anindicator showing how much interference these analogues would giveactually on the quantitative determination of DiAcSpm.

1. Urinary Polyamines

FIG. 1 provides graphs showing the results of analysis and quantitativedetermination of urinary polyamine levels by HPLC: these results aresummarized by component and classified into the three groups of healthypersons, patients with urogenital benign diseases, and patients withurogenital malignant tumors (e.g., prostate cancer, kidney cancer,testicular cancer). Usually, urinary polyamine levels are expressed ascreatinine-corrected values which are obtained from actually measuredvalues on random urine samples. Here, the mean of healthy persons wasobtained from the thus measured levels for individual components. Takingthe mean of healthy persons ±2S.D. as the cut-off value, ratios ofindividual components to the cut-off value are plotted in graphs. Forexample, as to DiAcSpd and DiAcSpm, healthy person levels of 0.30±0.11μmol/g creatinine (mean±S.D.) and 0.15±0.05 μmol/g creatinine,respectively, were obtained. Based on these levels, DiAcSpd 0.52 mol/gcreatinine and DiAcSpm 0.25 mol/g creatinine are taken as cut-offvalues. As shown in FIG. 1, with respect to acetylputrescine (AcPut),N¹- and N⁸-(mono)acetylspermidines (N¹-AcSpd and N⁸-AcSpd, which aremajor components present most abundantly in urine) and total polyamine,a large number of false negative results occur where even cancerpatients do not show high levels. Further, in many cases, tumor patientsand false positive benign disease patients cannot be discriminated withthose components. On the other hand, with respect to DiAcSpd andDiAcSpm, a large part of cancer patients shows high levels. Inparticular, DiAcSpm has remarkable characteristics; it is low in falsenegative ratio and very high in abnormal detection ratio. DiAcSpd ischaracterized by showing little increase in benign disease patients andthus being extremely low in false positive ratio (Sugimoto, M. et al.,J. Cancer Res. Clin. Oncol., 121, 317-319 (1995)). Not only inurogenital malignant tumors but also in colorectal cancer and breastcancer, urinary DiAcSpm in patients elevates at a high frequency.

This means that, if it is possible to develop a method of measuringdiacetylpolyamines easily and accurately, there would be a large demandfor them as novel tumor makers in clinical cancer diagnosis.

Table 1 below shows the results of analysis of various polyaminecomponents (diacetylpolyamines and their analogues) contained in theurine of healthy persons. TABLE 1 Urinary Polyamine Levels in HealthyPersons Mean¹⁾ Standard Deviation Coefficient of (μmol/g (S.D.)Variation Creatinine) (μmol/g Creatinine) (C.V.) (%) Total Polyamine22.2 6.1 27 Putrescine 0.54 0.52 97 Ac-Putrescine 9.57 3.52 37Cadaverine 0.23 0.24 105 Spermidine 0.17 0.14 80 Ac-Cadaverine 5.55 2.6448 N¹-Ac-Spermidine 2.70 0.84 31 N⁸-Ac-Spermidine 2.41 0.61 25DiAcSpermidine 0.301 0.109 36 Spermine 0.62 0.98 158 Ac-Spermine 0.0320.077 237 DiAcSpermine 0.107 0.047 46¹⁾Mean from 52 healthy persons

Major components in human urinary polyamines are variousmonoacetylpolyamines. DiAcSpm definitely belongs to trace components andit constitutes only 0.6% of the total polyamine. Monoacetylspermidines(N¹-AcSpd and N⁸-AcSpd) which are very similar to DiAcSpm in structureare found among the major components of urinary polyamines (FIG. 2) andusually, their contents reach 25-30 times the content of DiAcSpm. Giventhe similarity in structure, sufficient consideration should be made onthe possibility that anti-DiAcSpm antibody also cross-reacts with thesemonoacetylspermidines. In order to measure correctly the amount ofDiAcSpm in urine where monoacetylspermidines are present as interferingsubstances in an amount about 30-times greater than DiAcSpm, a specificantibody is necessary which strictly discriminates the substance ofinterest from interfering substances with low cross-reactivity.Therefore, it is demanded to prepare an antibody which shows suchspecificity and yet can accurately measure DiAcSpm of concentrationsaround 0.1 μM (which is the urinary DiAcSpm concentration of averagehealthy person).

2. Tumor Marker

In order to use the DiAcSpm of the present invention as a tumor marker,it is very important that the DiAcSpm contained in a sample should bedetected at a high sensitivity. As a means for such detection, anantibody to DiAcSpm may be used in the present invention.

As a method for purifying DiAcSpm-specific antibody, a method is knownin which rabbits are immunized with an Ac-Spermine derivative of bovineserum albumin, and then DiAcSpm-specific antibodies are purified fromthe resultant anti-sera (Hiramatsu, K. et al., J. Biochem., 124, 231-236(1998)). In the present invention, a DiAcSpm-specific antibody is newlyprepared based on that method, and a DiAcSpm-measuring kit comprisingthe antibody is prepared.

When hapten antibodies such as anti-DiAcSpm antibody are prepared, thedesign of the molecular structure of a hapten-carrier conjugate has alarge effect upon the performance of the resultant specific antibody. Itis reported that antibodies prepared using spermine bound to BSA withglutaraldehyde as a hapten show higher reactivity with spermine orspermidine than reactivity with acetylpolyamines in competitive ELISA.Therefore, in order to prepare an antibody that reacts preferentiallywith acetylpolyamines, it is indispensable that acylamide bond ispresent in the hapten-carrier conjugate.

When a polyclonal anti-serum shows about 5-6% cross-reactivity withN¹-AcSpd, such anti-serum cannot be used immediately for the purpose ofquantitative determination of DiAcSpm. In this case, a highly specificantibody component contained in the anti-serum must be purified by somemethods. As techniques to purify such a component, designing of themolecular structure of the ligand for an affinity resin is believed tobe important. Briefly, it is effective to create a structure similar toacetylpolyamines by allowing to form an acylamide bond at the bindingsite between a polyamine ligand and a resin, as in the design of theimmunizing antigen. For example, when carboxy-Toyopearl is derivatizedwith N⁸-AcSpd, Spd and AcSpm, affinity resins with affinity ligands verysimilar to DiAcSpd, N¹-or N⁸-AcSpd, and DiAcSpm in structure,respectively, will be obtained. By purifying antibodies using theseaffinity resins and utilizing difference in affinity formonoacetylspermidine, as well as DiAcSpd and DiAcSpm, it is possible toobtain an antibody highly specific to DiAcSpm (Hiramatsu, K. et al., J.Biochem., 124, 231-236 (1998)).

By purifying from antibodies in a crude serum an anti-DiAcSpm antibodyhaving high specificity required for quantitative determination ofurinary diacetylpolyamine, the titer of the resultant antibody naturallydecreases to a considerable extent. However, in the case of theabove-mentioned anti-serum, the titer of this purified antibody isconsidered to be sufficiently high for use in quantitative determinationof DiAcSpm by standard competitive enzyme immunoassay.

In order to accurately determine DiAcSpm contained in a trace amount inurine samples where substances with similar structures represented bymonoacetylspermidine are present abundantly by enzyme immunoassay,avoiding interferences by those substances, it is most important toobtain an antibody showing high specificity to DiAcSpm for establishmentof a measuring kit. The present invention has established technologiesfor developing DiAcSpm-specific antibodies and developing kits formeasuring urinary. DiAcSpm, and provides a method for utilizing DiAcSpmas a tumor marker.

3. Preparation of Antigen

Since DiAcSpm is an alkylamine of a low molecular weight, immunizingrabbits with DiAcSpm does not produce DiAcSpm-specific antibodies.Therefore, an immunizing antigen which has a number of DiAcSpm-mimickingstructures as side chains is prepared by binding AcSpm to bovine serumalbumin (a carrier protein) via acylamide bond.

In the present invention, an immunizing antigen may be prepared based onthe method of Kawakita et al. (Hiramatsu, K. et al., J. Biochem., 124,231-236 (1998)). First, the carrier protein BSA is reacted withS-acetylmercaptosuccinic acid to thereby prepareS-acetylmercaptosuccinic acid (AMS)-BSA complex as a reaction product.Further, by coupling AcSpm to AMS-BSA by acylamide bond via a divalentcrosslinking agent GMBS (N-(4-maleimidobutyryloxy) succinimide), animmunizing antigen AcSpm-GMB-BSA is prepared.

4. Preparation of Antibodies to DiAcSpm

In the present invention, the term “antibody” means any antibodymolecule (either polyclonal antibody or monoclonal antibody) capable ofbinding to DiAcSpm (antigen) or fragment thereof (e.g., Fab or F(ab′)₂fragment) or active fragment thereof having antigen-antibody reactionactivity (specifically, Fab, Fv, recombinant Fv, single chain Fv).

The antibody of the present invention (polyclonal antibody, monoclonalantibody and active fragment) may be prepared by any of various knownmethods. Methods for preparing such antibodies are well-known in theart.

Hereinbelow, the preparation of antibodies will be described morespecifically with reference to experiments and Examples. However, thepresent invention is not limited to these experiments and Examples.

(1) Preparation of Polyclonal Antibodies to DiAcSpm

An antigen prepared as described above is administered to a mammal. Themammal is not particularly limited. Rat, mouse, rabbit, or the like maybe used. Among them, rabbit is preferable.

Dose of the antigen per animal is 5-2 mg when adjuvant is not used and5-2 mg when adjuvant is used. Examples of adjuvants useful in theinvention include Freund's complete adjuvant (FCA), Freund's incompleteadjuvant (FIA) and aluminium hydroxide adjuvant. Immunization is carriedout mainly by intravenous, subcutaneous or intraperitoneal injection ofthe antigen. The immunization interval is not particularly limited.Immunization may be carried out at intervals of several days to severalweeks, preferably 2 to 5 weeks, 1 to 10 ten times, preferably 2 to 5times. Six to sixty days after the final immunization, antibody titersare measured by such methods as ELISA (enzyme-linked immunosorbentassay), EIA (enzyme immunoassay) or RIA (radioimmuno assay). Blood iscollected on the day when the maximum antibody titer is shown, and thenanti-serum is obtained.

Subsequently, reactivities with proteins of the polyclonal antibodies inthe resultant anti-serum are measured by methods such as ELISA, using aprotein such as BSA. As described earlier, DiAcSpm is present in a traceamount in a mixture comprising DiAcSpd, Spd, Spm, etc. Therefore, in thepresent invention, antibodies reactive with DiAcSpm are selected with astill higher accuracy.

Briefly, antibodies which show strong reactivity with DiAcSpm andsatisfy the following (i) and/or (ii) are selected: (i) cross-reactivitywith N¹-AcSpd is 0.1% or less; (ii) total interference on themeasurement results caused by cross-reaction with DiAcSpm analoguespresent in urine is 5% or less (preferably 3% or less).

(2) Preparation of Monoclonal Antibodies to DiAcSpm

(i) Collecting of Antibody-Producing Cells

An antigen prepared as described above is administered to a mammal(e.g., rat, mouse or rabbit). Dose of the antigen per animal is 500-200μg when adjuvant is not used and 500-200 μg when adjuvant is used.Examples of adjuvants useful in the invention include Freund's completeadjuvant (FCA), Freund's incomplete adjuvant (FIA) and aluminiumhydroxide adjuvant. Immunization is carried out mainly by intravenous,subcutaneous or intraperitoneal injection of the antigen. Theimmunization interval is not particularly limited. Immunization may becarried out at intervals of several days to several weeks, preferably 2to 5 weeks, 1 to 10 ten times, preferably 2 to 5 times. One to sixtydays after the final immunization, preferably 1-14 days thereafter,antibody-producing cells are collected. As antibody-producing cells,splenic cells, lymph node cells, peripheral blood cells, and the likemay be enumerated. Among all, splenic cells or local lymph node cellsare preferable.

(ii) Cell Fusion

In order to obtain hybridomas, cell fusion between antibody-producingcells and myeloma cells is performed. As the myeloma cell to be fused tothe antibody-producing cell, a commonly available cell strain derivedfrom an animal such as mouse may be used. A preferable cell strain to beused in the invention has drug selectivity, cannot survive in HATselection medium (containing hypoxanthine, aminopterin and thymidine) inunfused conditions, and can survive there only after fusion toantibody-producing cells. Examples of myeloma cells include mousemyeloma cell strains such as P3X63-Ag.8.U1(P3U1) or NS-I.

Subsequently, the above myeloma cell and the antibody-producing cell arefused. Cell fusion is carried out by mixing 1×10⁶ to 1×10⁷ cells/ml ofantibody-producing cells with 2×10⁵ to 2×10⁶ cells/ml of myeloma cellsin an animal cell culture medium (such as serum-free DMEM or RPMI-1640)(preferable cell ratio of antibody-producing cells to myeloma cells is5:1) in the presence of a cell fusion promoter. As the cell fusionpromoter, polyethylene glycol with an average molecular weight of1000-6000 daltons or the like may be used. Alternatively, it is alsopossible to fuse antibody-producing cells and myeloma cells in acommercial cell fusion device utilizing electric stimulation (e.g.,electroporation).

(iii) Selection and Cloning of Hybridomas

Hybridomas of interest are selected from the fused cells. Briefly, acell suspension is appropriately diluted with fetal bovineserum-containing RPMI-1640 medium or the like and then plated onmicrotiter plates. A selection medium is added to each well. Cells arecultured with appropriate exchange of the selection medium. As a result,cells growing approx. 14 days after the start of cultivation in theselection medium may be obtained as hybridoma cells.

Subsequently, whether or not antibodies reactive with DiAcSpm arepresent in the culture supernatant of proliferating hybridoma cells isscreened. The screening of hybridomas may be performed by conventionalmethods and is not particularly limited. For example, a part of theculture supernatant contained in a well in which hybridoma cells aregrowing may be collected and screened by enzyme immunoassay orradioimmunoassay.

The cloning of fused cells is performed by methods such as limitingdilution. Hybridomas producing such antibodies (as explained in thesubsection for polyclonal antibody) that show strong reactivity withDiAcSpm and satisfy one or both of the following properties (i) and (ii)are selected and established: (i) cross-reactivity with N¹-AcSpd is 0.1%or less; (ii) total interference on the measurement results caused bycross-reaction with DiAcSpm analogues present in urine is 5% or less(preferably 3% or less).

(iv) Collecting of Monoclonal Antibodies

As a method for collecting monoclonal antibodies from establishedhybridomas, a conventional cell culture method or abdominal dropsyformation method may be used.

In the cell culture method, hybridoma cells are cultured in an animalcell culture medium such as 10% fetal bovine serum-containing RPMI-1640medium, MEM medium or serum-free medium under conventional cultureconditions (e.g., 37° C., 5% CO₂ concentration) for 7 to 14 days. Then,antibodies are obtained from the culture supernatant.

In the abdominal dropsy formation method, approx. 1×10⁷ hybridoma cellsare administered into the abdominal cavity of an allogenic animal to themammal from which the myeloma cell derived, to thereby expand thehybridoma cells greatly. One to two weeks thereafter, the abdominaldropsy is collected.

When purification of antibodies is necessary in the above-describedmethod of collecting antibodies, antibodies may be purified byconventional methods such as ammonium sulfate salting out, ion exchangechromatography, gel filtration or affinity chromatography, or acombination of these methods.

5. Method of Detection of Tumors

Since DiAcSpm can be used as a clinical marker for cancer (tumormarker), it is possible to detect a tumor by reacting the antibody ofthe invention with a biological sample to thereby measure DiAcSpm in thebiological sample and using the measurement results as an indicator. Themeasurement of DiAcSpm may be performed by any of the conventionalhapten immunoassays and is not particularly limited. Examples of thetumor to be detected include, but are not limited to, the following.

-   (1) Oral Cavity, Nose and Throat

Tongue cancer, gingival cancer, malignant lymphoma, malignant blackcancer (melanoma), upper jaw cancer, nasal cavity cancer, laryngealcancer, pharyngeal cancer

-   (2) Cranial Nerve System

Glioma, meningioma

-   (3) Thyroid

Papillary adenocarcinoma of thyroid, follicular carcinoma of thyroid,medullary carcinoma of thyroid

-   (4) Respiratory Organs

Lung cancer (squamous carcinoma, adenocarcinoma, alveolar carcinoma,large cell undifferentiated carcinoma, small cell undifferentiatedcarcinoma, carcinoid)

-   (5) Breast

Breast cancer, breast Paget's disease, breast sarcoma

-   (6) Blood

Acute myelogenous leukemia, acute promyelocytic leukemia, acutemyelogenous monocytes leukemia, acute monocytes leukemia, acutelymphocytic leukemia, acute undifferentiated leukemia, chronicmyelogenous leukemia, chronic lymphocytic leukemia, adult T cellleukemia, malignant lymphoma (lymphosarcoma, reticulosarcoma, Hodgkin'sdisease), multiple myeloma, primary macroglobulinemia

-   (7) Digestive Organs

Esophagus cancer, gastric cancer, gastrointestinal malignant lymphoma,pancreatic cancer, biliary tract cancer, gallbladder cancer, duodenumcancer, colorectal cancer, liver cancer

-   (8) Female Genital Organs

Uterus cancer, ovary cancer, uterus sarcoma (leiomyosarcoma,rhabdomyosarcoma, lymphosarcoma, reticulosarcoma)

-   (9) Urinary Organs

Urinary tract malignant tumor (prostate cancer, kidney cancer, bladdercancer, testis tumor, urethral cancer)

-   (10) Locomotorium

Rhabdomyosarcoma, fibrosarcoma, osteosarcoma, chondrosarcoma, sarcoma ofsynovial membrane, mucous sarcoma, liposarcoma, Ewing's sarcoma,multiple myeloma

-   (11) Skin

Skin cancer, skin Bowen's disease, skin Paget's disease, skin melanoma

In the present invention, the cancer to be detected and evaluated may beone selected from the above or a complication of two or more cancers.Preferably, the target cancer is at least one selected from the groupconsisting of colorectal cancer, urogenital malignant tumors (e.g.prostate cancer, kidney cancer, bladder cancer, testicular cancer,urethral cancer), breast cancer, pancreatic cancer, biliary tractcancer, lung cancer, liver cancer, uterine cervix cancer, brain tumor,myelogenous leukemia and malignant lymphoma.

Examples of methods for detecting tumors using the antibody of theinvention will be described later, but use of the antibody is notparticularly limited to those Examples.

Biological samples are taken from patients suspicious of cancer andsubjects of health examination, followed by preparation of samples forDiAcSpm measurement. Examples of the biological sample useful in theinvention include blood, urine and tissue. Urine is preferable becauseit is easy to handle and imposes less burden on patients.

Subsequently, the thus prepared sample for measurement is reacted withthe above-described antibody. Measurement of DiAcSpm may be performed byconventional ELISA. First, microplates are coated with the antigen(DiAcSpm) in advance. On the other hand, DiAcSpm in the biologicalsample and DiAcSpm in the standard solution are pre-reacted with theanti-DiAcSpm specific antibody, and the resultant reaction solutions areplated on the microplates. The antibody remaining unreacted binds to theDiAcSpm on the microplate. Then, HRP-labeled anti-rabbit IgG antibody(which is a secondary antibody) is added to the microplate for reaction.Finally, the amount of DiAcSpm contained in the biological sample isdetermined by the color development reaction catalyzed by HRP.

6. Evaluation of Tumors

The state of the tumor is evaluated or diagnosed using, as an indicator,the detection results obtained by the detection method described inabove subsection 5. Detection results exceeding the specific cut-offvalue are classified as DiAcSpm positive, and detection results belowthe specific cut-off value are classified as DiAcSpm negative. When theresult is positive, it is judged that the relevant patient or subjectmay have cancer, and then the state of the tumor can be evaluated.

The state of a tumor means the presence or absence of the tumor or theprogression thereof. Specifically, the presence or absence of canceroccurrence, the progression of cancer, the degree of the malignancy ofcancer, the presence or absence of cancer metastasis, the presence orabsence of cancer recurrence, and the like may be enumerated. In theevaluation of the above-mentioned state, one state may be selected.Alternatively, a plurality of states may be selected in an appropriatecombination. In order to evaluate the presence or absence of cancer,whether the relevant patient has developed cancer or not is judged. Thedegree of the malignancy of cancer can be an indicator showing how muchadvanced the cancer is. This degree may be evaluated by staging thecancer or classifiing the cancer into the so-called early cancer oradvanced cancer. Cancer metastasis is evaluated by whether neoplasms areoccurring at distant sites from the primary lesion. Recurrence is judgedby whether or not the cancer appeared again after intermission orremission.

In the present invention, in the case of colorectal cancer for example,the cancer can be detected and evaluated even at stage 0 or I (earlycancer) in the same manner as the cancer at stage II to IV (advancedcancer). In the case of breast cancer, the cancer can be detected andevaluated even at stage I or II (early cancer) in the same manner as thecancer at stage III to IV (advanced cancer).

7. Reagent and Kit for Tumor Detection Comprising the Antibody of theInvention

In the present invention, it is possible to use an antibody to DiAcSpmas a reagent for tumor detection.

Conventionally, when polyamines are measured in general biochemicaltests, urinary polyamines were measured collectively as a single classof components. Relations between each of those similar structures anddisease conditions have hardly been examined. Then, a method has beenestablished in which amounts of urinary polyamines are measureddiscretely. It has been confirmed that, in particular, DiAcSpm (onespecies of polyamine) is highly elevated at the time of occurrence andat the time of recurrence after treatment of prostate cancer orcolorectal cancer. This means that, if it is possible to develop amethod of measuring diacetylpolyamines easily and accurately, therewould be a large demand for them as novel tumor makers in clinicalcancer diagnosis.

In conventional measuring methods, immunological techniques which enablethe handling of a great number of samples with cheap measuringinstruments are employed. A simple measuring method using a polyclonalantibody has already been known (Hiramatsu, K. et al., J. Biochem., 124,231-236 (1998)). However, an ELISA measuring system has not been putinto a kit; also, mass production of DiAcSpm (a most important componentin the measuring system) has not been achieved.

In the present invention, the inventors contemplated a system formeasuring urinary DiAcSpm by competitive ELISA using DiAcSpm antibody,based on Kawakita et al.'s simple immunological measuring method as amodel. As a solid phase antigen, monoacetylspermine coupled to a peptideby acylamide bond and having a DiAcSpm-mimicking structure(AcSpm-HMC-peptide) may be used. This antigen is obtained by couplingmonoacetylspermine to a water-soluble peptide by acylamide bond viabivalent crosslinking reagent (HMCS) (Hiramatsu, K. et al., J. Biochem.,124, 231-236 (1998)).

The DiAcSpm measuring kit of the invention must be capable of measuringDiAcSpm with high sensitivity. Further, this kit is required to havereproducibility if it is intended to be used in clinical scenes ofcancer diagnosis. Upon such a premise, the present invention aimed atestablishment of a measuring system for DiAcSpm as a diagnosis marker.

The DiAcSpm measurement ELISA kit of the invention is capable of settingits standard region ranging from 4.53 to 145 nM by adjusting the solidphase antigen concentration toward the low concentration side. The solidphase antigen concentration is 1-0.1 μg/ml, preferably 0.07 μg/ml. As aresult, sensitivity and measuring accuracy sufficient to measure urinaryDiAcSpm can be achieved.

Measurement accuracy is an indicator which shows to what extentindividual measured values would vary when one assay has been performedusing one same sample aliquoted into a plurality of test tubes or wells.Statistically, measurement accuracy is expressed as coefficient ofvariation (CV), that is, ratio (%) of standard deviation to mean. In thepresent invention, this CV is referred to as reproducibility.Reproducibility is preferably 10% or below, more preferably 5% or below.

The kit of the present invention has the following performances: lowerdetection limit in actual measurement: 9.06 nM that corresponds to alower detection limit in urine or other sample of 36.2 nM (9.06 nM×4).Within-run reproducibility is 10% or less, preferably around 5%.Between-day reproducibility is 10% or less, preferably about 8-10%. Inboth reproducibilities, CV is 10% or less. With respect to the influenceof co-existing substances, conjugated bilirubin, glucose, hemoglobin andascorbic acid do not give any influence upon DiAcSpm measurement.

The kit of the invention may also contain antigen-coated microplates,DiAcSpm standard product (STD), antibody dilutions, HRP-labeledanti-rabbit IgG antibody, OPD (ortho-phenylene-diamine) tablets,substrate solution, reaction termination solution, concentrated washingsolution or the like selected appropriately, in addition to the antibodyof the invention.

Hereinbelow, the present invention will be described more specificallywith reference to the following Examples. However, the present inventionis not limited to these Examples.

EXAMPLE 1

Preparation of Antibodies

(1) Preparation of Immunizing Antigen and Immunization

In this Example, the inventors examine more efficient preparation ofanti-DiAcSpm antibodies in order to put into practical use the techniqueof extracting specific components from polyclonal antibodies by affinitypurification to practical use in the production of anti-DiAcSpmpolyclonal antibodies. For putting into practice use the technique ofextracting specific components from polyclonal antibodies by affinitypurification, extremely important things are (i) to make it clear ifanti-DiAcSpm antibodies showing high specificity can be obtainedreproducibly and (ii) to ascertain at what frequency mammals producingsuch antibodies can be obtained when mammals are immunized with one sameantigen. Then, the present inventors have paid attention to thefollowing three points in examining the efficiency and reproducibilityof antibody production toward industrial production of measuring kits.

-   (a) The probability that antibody titers rise is low because the    antigen of interest is a low molecular weight molecule.-   (b) Antibody titer rising ratios vary depending on the species of    rabbit to be immunized.-   (c) Antibody titer rising ratios vary depending on the lot of the    immunizing antigen.

In order to examine the condition (a) described above, eight rabbits ofJapanese White Rabbit species (male: standard 2.5-3.0 kg, hereinafterabbreviated to “JPW”) were immunized with immunizing antigen Lot 1 undersame conditions. Then, whether antibody titer rising ratios vary amongindividual rabbits or not was examined.

In order to examine the condition (b) described above, nine rabbits ofNew Zealand White Rabbit species (male: standard 2.5-3.0 kg, hereinafterabbreviated to “NZW”) and ten rabbits of Japanese white Rabbit species(male: standard 2.5-3.0 kg, hereinafter abbreviated to “JPW”) werechosen as animal species to be immunized. Then, whether antibody titerrising ratios vary between animal species was examined.

In order to examine the condition (c) described above, five differentlots of immunizing antigen AcSpm-GMB-BSA were newly prepared. Then,whether antibody titer rising ratios vary among individual animalsimmunized with different lots was examined.

The immunizing antigen was prepared as described below, based on themethod of Kawakita et al. (Hiramatsu, K. et al., J. Biochem., 124,231-236 (1998)). Since DiAcSpm is an alkylamine of a low molecularweight, it is impossible to obtain DiAcSpm-specific antibodies byimmunizing rabbits with DiAcSpm itself. Therefore, an immunizingantigen, AcSpm-GMB-BSA, which has a number of DiAcSpm-mimickingstructures as side chains was prepared by coupling AcSpm to bovine serumalbumin (hereinafter abbreviated to “BSA”) (a carrier protein) viaacylaride bond. Briefly, BSA which functions as a carrier protein wasreacted with S-acetylmercaptosuccinic acid to therein obtain thereaction product S-acetylmercaptosuccinylated BSA (hereinafterabbreviated to AMS-BSA). Further, AcSpm was coupled to AMS-BSA byacylamide bond via a bivalent crosslinking reagent GMBS to therebyobtain the immunizing antigen AcSpm-GMB-BSA.

Immunization was carried out by injecting subcutaneously in the back ofanimals an emulsion prepared by mixing equal volumes of the immunizingantigen and adjuvant (for first immunization: complete adjuvant; forbooster immunization: incomplete adjuvant). Immunizations were carriedout at the intervals of two weeks at doses of 1 mg/animal for the firstimmunization and 0.3 mg/animal for booster immunizations. Seven daysafter the third immunization, partial exsanguination was carried out,followed by checking of the antibody titers in antisera by ELISA (Table2).

Antibody titers are shown using the antiserum obtained by Kawakita etal. as a standard. Specifically, the reactivity of each rabbit antiserumis expressed as a percentage taking the reactivity of the Kawakita etal.'s antiserum at a dilution of 27,000-fold as 100.

As a result, the rise of antibody titer was low in JPW1-JPW8 and NZW1immunized with antigen Lot No. 1; the highest antibody titer shown inthese rabbits was 12.5% in JPW7. On the other hand, high antibody titersof 69.1% (JPW9) and 88.1% (NZW2) could be obtained in both speciesimmunized with antigen Lot No. 2.

The results of this experiment revealed that there is difference in therise of antibody titers depending on the lot of the antigen. However,the checking of antibody titers using the partially exsanguinatedsamples revealed no remarkable difference in the rise of antibody titersamong rabbit individuals or between species. TABLE 2 Immunizing Antigenand Antibody Titer in Antiserum Immunizing Code No. Antigen Lot AntibodyTiter (%) in Antiserum JPW1 No. 1 4.71 JPW2 No. 1 3.73 JPW3 No. 1 6.52JPW4 No. 1 6.98 JPW5 No. 1 3.20 JPW6 No. 1 4.29 JPW7 No. 1 12.5 JPW8 No.1 9.09 JPW9 No. 2 69.1 JPW10 No. 3 6.86 NZW1 No. 1 7.59 NZW2 No. 2 88.1NZW3 No. 3 5.76 NZW4 No. 4 28.4 NZW5 No. 5 7.11 NZW6 No. 6 7.41 NZW7 No.6 9.70 NZW8 No. 7 27.8 NZW9 No. 7 34.0

(2) Purification of Anti-DiAcSpm Polyclonal Antibodies

Among the antisera obtained from 19 rabbits, three anti-DiAcSpmpolyclonal antisera (JPW9, NZW2 and NZW9) showed high antibody titers.Step-wise affinity purification was carried out to examine whetherspecific antibodies with sufficient purity applicable to a urinaryDiAcSpm measuring system can be obtained. The affmity purification wascarried out step-wise based on the method of Kawakita et al. (Hiramatsu,K. et al., J. Biochem., 124, 231-236 (1998)), using DiAcSpm column andDiSpd column prepared by derivatizing carboxy-Toyopearl with AcSpm andN8-AcSpd (FIG. 3).

The results revealed that antiserum NZW2 contained an antibody whosecross-reactivity with N¹-AcSpd (which is present in urine as a DiAcSpmanalogue in an amount about 30-times greater than DiAcSpm) is 0.1% orless. Further, the total interference on the measurement results causedby the cross-reaction of this antibody with DiAcSpm analogues present inurine was 2.9%; thus, cross-reactivity of 5% or less was achieved (FIG.4, Table 3). TABLE 3 Cross-Reactivity of Anti-DiAcSpm Code No. NZW 2DiAcSpm AcSpm N1-AcSpd DiAcSpd N8-AcSpd AcPut κi_(DiAcSpm)/κi_(“s”) (%). . . (1) 100 0.418 0.100 0.032 0.001 — Ratio of DiAcSpm in urine . . .(2) 1.00 0.314 26.5 2.95 23.7 93.8 Cross-reactivity (1) × (2)% 100 0.1312.65 0.094 0.024 — Tortal  2.90%

Thus, it was possible to prepare an anti-DiAcSpm specific antibody withhigh specificity applicable to the measurement of urinary DiAcSpm byimmunizing rabbits with a newly prepared immunizing antigenAcSpm-GMB-BSA. This proved the efficacy of the immunizing antigen havingDiAcSpm-mimicking structures as side chains, which Kawakita et al. haveinvented in order to obtain anti-DiAcSpm specific antibodies.

EXAMPLE 2

Measurement of DiAcSpm Using the Kit

1. Establishment of Measuring Parts and Basic Data of ELISA MeasuringSystem

As a measuring method, competitive ELISA using a solid phase antigenAcSpm-HMC-peptide was performed based on the method of Kawakita et al.(Hiramatsu, K. et al., J. Biochem., 124, 231-236 (1998)). As the primaryantibody, the anti-DiAcSpm specific antibody prepared in Example 1 wasused. As the secondary antibody, a commercial, goat-derived HRP-labeledanti-rabbit IgG antibody (Bio-Rad) was used. The concentration of thesolid phase antigen was selected as follows. Since the normal urinaryDiAcSpm level is approx. 100 nM, a range of authentic DiAcSpmconcentrations from 6.25 nM to 200 nM was chosen. Then, a concentrationof the solid phase antigen (0.07 μg/ml) which yields the most idealisticcompetitive curve with DiAcSpm in this concentration range was selected.With respect to the concentration of the anti-DiAcSpm antibody, 0.02μg/ml was selected so that 50% of the maximum reaction efficiency isachieved when the concentration of the solid phase antigen is 0.07μg/ml.

With respect to the HRP-labeled anti-rabbit IgG (secondary antibody),its sensitivity was examined in a range from 2000-fold to 5000-folddilution. As a result, the highest sensitivity was obtained at 2000-folddilution. Therefore, HRP-labeled anti-rabbit IgG 2000-fold dilution wasselected as the condition of the secondary antibody.

Further, in order to evaluate the accuracy and performance of theDiAcSpm measurement ELISA, within-day reproducibility and between-dayreproducibility were obtained using two different control samples(called Sample A and Sample B) and evaluated (N=20). Further, inaddition-recovery tests, DiAcSpm standard product of a knownconcentration was added to the urine of healthy persons, followed bydetermination of the recovery ratio. In order to evaluate theperformance of the kit, dilution tests were also performed on threeurine samples with different DiAcSpm concentrations.

In order to compare HPLC (which has already been established as a methodfor determining urinary DiAcSpm) with the kit of the present invention,correlation was examined on 30 samples. Further, the influence ofconjugated bilirubin, glucose, hemoglobin and ascorbic acid (which areco-existing substances in urine) upon the measurement system wasconfirmed.

2. Results

With respect to the performances of the assay system according to thekit of the invention, the standard curve showed linearity in the rangefrom 6.25 to 200 nM, and both within-day and between-dayreproducibilities were good (CV=10% or less). In the dilution test usingthree urine samples with different DiAcSpm concentrations, a gooddilution curve was obtained for every urine sample as a result ofmeasurement of serially diluted samples on each of the three (FIGS. 5 to7).

In the addition-recovery test, 4-fold or more dilution of urine yieldedgood results of addition-recovery ratios 96.3-108% without individualdifference. Therefore, it was decided to begin the dilution of urea at4-fold or more.

The performances of the kit of the invention were as follows: actuallymeasured minimum detection value: 12.5 nM; detection sensitivity insample measurement: 50 nM (12.5 nM×4). The within-run reproducibilitywas CV=4.87% on Sample A and CV=5.20% on Sample B. The between-dayreproducibility was CV=7.53% on Sample A and CV=9.46% on Sample B.

Therefore, it was confirmed that both reproducibilities satisfied thetarget value of 10% or less on each of the Samples (Table 4). Withrespect to co-existing substances, it was confirmed that conjugatedbilirubin (10 mg/dL or less), glucose (1000 mg/dL or less), hemoglobin(400 mg/dL or less) and ascorbic acid (100 mg/dL or less) give noinfluence upon DiAcSpm measurement at the indicated concentrations.

Further, in order to evaluate the ELISA kit for measuring DiAcSpm, urinesamples measured by HPLC which serve as reference were measured by theELISA kit, and then the results obtained from both methods were comparedand examined. As a result, correlation with HPLC was Y(nM)=1.01X+73.2,R²=0.978. This is a very good result. From these results, it wasdemonstrated that the DiAcSpm measurement kit of the invention iscapable of measuring urinary DiAcSpm with high accuracy.

The details of basic data obtained in these experiments are shown inTable 4 and FIGS. 5 to 8. TABLE 4 Within-day Between-day reproducibility(N = 20) reproducibility (N = 20) Sample DiAcSpm conc. (nM) SampleDiAcSpm conc. (nM) No. A B No. A B 1 94.0 34.5 1 102 37.7 2 89.0 33.0 2104 29.9 3 95.3 35.2 3 100 31.0 4 97.9 34.3 4 110 33.1 5 103 32.4 5 90.629.0 6 96.6 32.1 6 94.7 31.3 7 89.8 31.4 7 95.5 32.2 8 91.6 32.5 8 92.729.5 9 89.8 30.8 9 110 39.3 10 104 30.9 10 108 37.7 11 96.9 31.5 11 94.535.6 12 99.5 34.4 12 104 32.6 13 94.6 30.8 13 106 39.3 14 103 32.1 14115 32.5 15 93.1 29.9 15 93.1 32.1 16 91.9 31.5 16 89.2 31.6 17 95.029.7 17 100 36.2 18 92.8 33.3 18 113 36.9 19 100 32.0 19 99.3 33.7 2091.3 35.3 20 96.8 34.0 mean 95.5 32.4 mean 101 33.8 SD 4.65 1.68 SD 7.63.19 CV (%) 4.87 5.20 CV (%) 7.53 9.46

EXAMPLE 3

Detection of Colorectal Cancer

Relations between stages of colorectal cancer (according to “JapaneseClassification of Colorectal Carcinoma” by Japanese Society for Cancerof the Colon and Rectum) and urinary DiAcSpm levels(creatinine-corrected values of concentrations in random urine) wereexamined on 250 patients with colorectal cancer. The cut-off value ofDiAcSpm was set at 0.25 μmol/g creatinine (mean of healthy persons+2SD),and levels exceeding this were regarded as positive and levels belowthis were regarded as negative. As a result, out of 250 cases examined,DiAcSpm positive cases were 185 (74.0%); CEA positive cases were 94(37.6%); and CA-19-9 positive cases were 36 (14.4%) (Tables 5 to 7).Thus, compared with existing tumor markers, DiAcSpm exhibited remarkablyhigh detection sensitivity. Further, examination of relations betweenstages of patients and positive ratios based on findings obtained bypathological tests revealed that urinary DiAcSpm shows high positiveratios in both early and advanced cancers, as shown in Table 5. Thisdemonstrates that DiAcSpm levels in the urine of colorectal cancerpatients rise at high frequency even at the stages of early cancer.TABLE 5 Positive Ratio of Urinary DiAcSpm by Stage of Colrectal CancerPositive Negative Positive Ratio % stage 0 13 8 62 Early staged stage I24 16 60 cancer stage II 42 18 70 Advanced stage III 92 21 81 cancerstage IV 14 2 88 Total 185 65

In Table 5, “Stage 0” means the depth of invasion is m. In other words,lesions are limited to the mucosal layer of the intestinal tract. “StageI” means that the degree of invasion is up to the submucosal layer (sm)or the proper muscle layer (mp) and that the cancer is n₀ tumor (with nolymph node metastasis). “Stage 0” and “Stage I” are classified intoearly staged cancer. If cancer is found at these stages, it isestablished clinically that good prognosis is expected in every case.

Further, for the purpose of comparison, relations between serum CEA orserum CA19-9 levels (now used clinically) and stages of colorectalcancer were examined on the same group of patients as examined above.With respect to CEA, the standard value was set at 5 ng/ml and levelsexceeding this were regarded as positive and levels below this wereregarded as negative. With respect to serum CA19-9, the standard valuewas set at 37 U/ml and levels exceeding this were regarded as positiveand levels below this were regarded as negative.

As a result, as shown in Tables 6 and 7, positive ratios were low inearly cancer in both serum CEA and serum CA19-9. TABLE 6 Positive Ratioof Serum CEA by Stage of Colorectal Cancer Positive Negative PositiveRatio % Stage 0 2 19 9.5 Early staged Stage I 4 36 10 cancer Stage II 2535 42 Advanced Stage III 53 60 47 cancer Stage IV 10 6 63 Total 94 156

TABLE 7 Positive Ratio of Serum CA19-9 by Stage of Large Bowel CancerPositive Negative Positive Ratio % stage 0 1 20 4.8 Early staged stage I3 37 7.5 cancer stage II 3 57 5.0 Advanced stage III 24 89 21 cancerstage IV 5 11 30 Total 36 214

While the positive ratio of urinary DiAcSpm for early staged colorectalcancer was about 60%, the positive ratios of serum CEA and serum CA19-9were about 10% and about 5-8%, respectively. This demonstrates thatDiAcSpm is by far superior in clinical diagnosis of colorectal cancer tothe currently used tumor markers CEA and CA19-19, compared to theirpositive ratios for early staged large bowel cancer. Therefore, by usingurinary diacetylspermine as a tumor marker and detecting thediacetylspermine with the antibody of the invention, it becomes possibleto detect colorectal cancer with still higher sensitivity and at earlystages. Start of treatment at early stages will increase the ratio ofcompletely cured cases. Thus, use of this marker can greatly contributeto the clinical diagnosis of colorectal cancer.

Further, by adding urinary diacetylspermine as one of health examinationitems, it is possible to make early diagnosis of digestive organ cancersincluding colorectal cancer more sensitive and accurate by far than thediagnosis currently done.

EXAMPLE 4

Detection of Breast Cancer

Urinary diacetylspermine levels were measured on 17 patients with breastcancer to thereby obtain the following results. When the cut-off valueof diacetylspermine excreted in urine is set at 0.25 μmol/g creatinine(mean of healthy persons +2S.D.), the urinary diacetylspermine level wasexceeding the cut-of value in every patient. In particular, 9 patientsout of 17 showed high levels more than two times the standard value(FIG. 9). Urinary diacetylspermine has a higher positive ratio thanCA15-3 that is frequently used as a tumor marker for breast cancer. Thisdemonstrates that urinary diacetylspermine is useful as a tumor markerfor detecting breast cancer with high sensitivity.

EXAMPLE 5

Detection of Pancreatic/Biliary Tract Cancers

Cancer detection was carried out on 125 patients with pancreatic/biliarytract diseases. Patients with pancreatic endocrine tumor, hepatocellularcarcinoma, acute inflammations (such as cholecystitis or pancreatitis)and postoperative patients who have undergone an operation within lastthree months were excluded from the target patients.

The particulars of the target patients are as follows. Male: 70 cases;female: 55 cases; age: 28-86 (63.5±12.2)

Of these target patients, 52 cases were preoperative or postoperativepatients with benign diseases (control); 22 cases were preoperativepatients with malignant diseases; and 51 cases were postoperativepatients with malignant diseases (10 patients have recurrence).

Of the above, the number of adenocarcinoma cases was 32 and the numberof adenoma cases was 8.

Urinary diacetylspermine, serum CEA (high sensitivity, 2.5 ng/ml) andCA19.9 (high specificity, 37 U/ml) were detected by ELISA.

After cut-off values of urinary diacetylspermine were set, relationsbetween the diagnosis of benign/malignant and urinary/serum tumormarkers; relations between the diagnosis of recurrence and urinary/serumtumor markers; relations between stages and urinary/serum tumor markers;and relations between excision/non-excision and urinary/serum tumormarkers were examined.

The results are shown in Tables 8 to 11 and FIGS. 10 to 15. TABLE 8Relations between Pancreatic/Biliary Tract Diseases and Urinary DiAcSpm,Serum CEA or CA19-9 Benign Adenoma Adenocarcinoma Urinary 267.2 ±143.0^($) 243.2 ± 96.1 621.5 ± 584.0^($) DiAcSpm (nmol/g Creatinine)Serum CEA (ng/ml)  1.2 ± 0.8  1.4 ± 1.2  39.2 ± 158.5 CA19-9  16.8 ±21.3^(#)  9.0 ± 5.07 825.2 ± 23,996.8^(#)^($)p < 0.001,^(#)P = 0.0209

TABLE 9 Relations between Benign/Malignant and Urinary DiAcSpm, SerumCEA or CA19-9 DiAcSpm CEA CA19-9 Positive Negative Positive NegativePositive Negative Malignant 22 10 14 18 21 11 Benign 8 44 4 48 7 45Sensitivity  75%^(a)  44%^(a) 75% Specificity 81% 92% 80% Positivepredictive value 71% 78% 86% Negative predictive value 84% 73% 87%Efficacy 79% 74% 79%^(a)P = 0.044

TABLE 10 Relations between Recurrence and Urinary DiAcSpm, Serum CEA orCA19-9 DiAcSpm CEA CA19-9 Positive Negative Positive Negative PositiveNegative Recurrence Present 8 2 7 3 7 3 None 14 27 9 32 7 34 Sensitivity80% 70% 70% Specificity 66% 78% 83% Positive predictive value 36% 44%50% Negative predictive value 93% 91% 92% Efficacy 69% 76% 80%

TABLE 11 Relations between Tumor Stages and Urinary DiAcSpm, Serum CEAor CA19-9 DiAcSpm CEA CA19-9 Stage Positive Negative Positive NegativePositive Negative IIb 3 3 0 6 4 2 III 2 0 1 1 1 1 IV 19 5 11 13 16 8

As shown in above Tables 8 to 11 and FIGS. 10 to 15, urinarydiacetylspermine shows high sensitivity against pancreatic/biliary tractcancers almost equivalent to that of serum CA19-9 which is regarded as ahighly sensitive tumor marker. Urinary diacetylspernine, which does notrequired collection of blood samples, can be said a “universal” tumormarker without organ specificity. Besides, urinary diacetylspermine is atumor marker useful in mass-screening and examination of high riskgroups.

Even at Stage IIB (a relatively early stage), urinary diacetylspermineindicated positive in 50%, which demonstrates that urinarydiacetylspermine was also effective in detecting earlypancreatic/biliary tract cancers.

EXAMPLE 6

Measurement of Diacetylspermine by Stage of Breast Cancer

In this Example, relations between stages of breast cancer and urinaryDiAcSpm levels (creatinine-corrected values of concentrations in randomurine) were examined on 83 patients with breast cancer. Stages of breastcancer and contents thereof are as described in Table 12. TABLE 12Stages of Breast Cancer (based on TNM classification) Stage Contents 0Non-invasive cancer or Paget's disease with no tumor. I Tumor 2 cm orless in size, and no evidence of metastasis to lymph nodes under thearm. IIA Tumor 2 cm or less in size, and evidence of metastasis to lymphnodes under the arm. Tumor more than 2 cm but not more than 5 cm, and noevidence of metastasis to lymph nodes under the arm. IIB Tumor more than2 cm but not more than 5 cm, and evidence of metastasis to lymph nodesunder the arm. Tumor more than 5 cm, and no evidence of metastasis tolymph nodes under the arm. IIIA Tumor more than 5 cm, and evidence ofmetastasis to lymph nodes under the arm. Regardless of the size oftumor, metastases to the under arm lymph nodes fixed to one another orto other structures are observed. IIIB Tumor fixed on the chest wallwith no movement; edema or ulceration of the skin or satellite skinnodules are observed. Evidence of metastases to parasternal lymph nodes(lymph nodes around the artery behind the sternum). Breast canceraccompanied with redness, edema, pain, etc. like in mastitis(inflammatory breast cancer); easily mistaken as inflammation. IVEvidence of metastases to supraclavicular lymph nodes. Accompanies withmetastases to other distant organs (distant organs such as bone, lung orliver, and distant lymph nodes other than supraclavicular lymph nodes)Stages 0 to IIIB have no distant metastasis.

Of the above-mentioned breast cancer patients, 15 patients were at stageI; 15 patients were at stage II; 4 patients were at stage III; and 47patients were at stage IV. The cut-off value of DiAcSpm was set at 0.25μmol/g creatinine (mean of healthy persons +2SD). DiAcSpm levelsexceeding this were regarded as positive, and DiAcSpm levels below thiswere regarded as negative.

The results are shown in Table 13. TABLE 13 Positive Ratio of UrinaryDiAcSpm by Stage of Breast Cancer Positive Negative Positive Ratio %stage I 3 12 20 stage II 6 11 35 stage III 3 1 75 stage IV 38 9 81 Total50 33

Relations between the stage of breast cancer and serum CEA levels wereexamined on the same group of patients as examined above. The standardvalue of CEA was set at 5 ng/ml, and levels exceeding this were regardedas positive, and levels below this were regarded as negative.

The results are shown in Table 14. TABLE 14 Positive Ratio of Serum CEAby Stage of Breast Cancer Positive Negative Positive Ratio % stage I 015 0 stage II 1 16 6 stage III 2 2 50 stage IV 28 19 60 Total 31 52

Relations between the stage of breast cancer and serum CA15-3 levelswere examined on the same group of patients as examined above. Thestandard value of CA15-3 was set at 23 U/ml, and levels exceeding thiswere regarded as positive, and levels below this were regarded asnegative.

The results are shown in Table 15. TABLE 15 Positive Ratio of SerumCA15-3 by Stage of Breast Cancer Positive Negative Positive Ratio %stage I 0 15 0 stage II 0 17 0 stage III 2 2 50 stage IV 29 18 62 Total31 52

As shown in above Tables 13 to 15, at any stage of early cancer andadvanced cancer, the urinary DiAcSpm level in the urine of breast cancerpatients showed a high positive ratio. In particular, the positiveratios of 20-35% at stage I and stage II are comparable to the positiveratios obtained by examination with PET (positron emission tomography).The obtained DiAcSpm levels, CEA levels and CA15-3 levels were comparedat the relatively early stage (a group combining stages I and II) andthe advanced cancer stage (a group combining stages III and IV).

The results are shown in Table 16. TABLE 16 Positive Ratios of DiAcSpmand Tumor Markers at Early and Advanced Stages of Breast Cancer DiAcSpmCEA CA 15-3 Posi- Nega- Positive Posi- Nega- Positive Posi- Nega-Positive Stage n tive tive ratio (%) tive tive ratio (%) tive tive ratio(%)  I + II 32 9 23 28.1 1 31 3.1 0 32 0 III + IV 51 41 10 80.3 30 2158.8 31 20 60.8 Total 83 50 33 60.2 31 52 37.3 31 52 37.3

As shown in Table 16, the respective positive ratios at the relativelyearly stage were 28.1% in DiAcSpm level, 3.1% in CEA level, and 0% inCA15-3 level. Significant difference was observed both between DiAcSpmlevel and CEA level (p=0.0064) and between DiAcSpm level and CA15-3level (p=0.0010). Similarly, the respective positive ratios at theadvanced stage were 80.3% in DiAcSpm level, 58.8% in CEA level, and60.8% in CA15-3 level. Significant difference was observed both betweenDiAcSpm level and CEA level (p=0.018) and between DiAcSpm level andCA15-3 level (p=0.030). As a whole, the respective positive ratios were60.2% in DiAcSpm level and 37.3% in both CEA level and CA15-3 level.Significant difference was observed between DiAcSpm level and either CEAor CA15-3 level with p=0.0032. Therefore, it was demonstrated thatDiAcSpm detects breast cancer with by far higher sensitivity, evencompared with existing tumor markers CEA and CA 15-3 now usedclinically.

EXAMPLE 7

Detection of Various Cancers

In this Example, cancers in the pancreas/biliary tract, lung, liver,uterus and blood were detected. The cut-off value was set at 325 (nmol/gcreatinine). With respect to pancreatic cancer and biliary tract cancer,test samples different from those in Example 5 were used.

The results are shown in Table 17. TABLE 17 Number of positive Cancerspecies samples Positive ratio (%) Pancreatic cancer, Biliary tract39/53 74 cancer Lung cancer 29/35 83 Liver cancer 31/49 63 Uterinecervix cancer 10/15 67 Myelogenous leukemia 4/6 67

As shown in Table 17, with the use of the kit of the invention, it waspossible to detect not only colorectal cancer and breast cancer but alsolung cancer, liver cancer, uterine cervix cancer and myelogenousleukemia (chronic and acute) with high positive ratios.

EXAMPLE 8

Detection of Various Brain Tumors

In this Example, positive ratios of urinary DiAcSpm in various braintumors were measured. The results are shown in Table 18. TABLE 18 Numberof positive Cancer species samples Positive ratio (%) Metastatic braintumor 30/33 90.9 Grade 2 glioma 1/2 50 Grade 3 glioma 6/8 75 Grade 4glioma 4/4 100 Glioma overall 11/14 78.6 Primary malignant lymphoma of3/3 100 central nerve system

The positive ratio of metastatic brain tumor was fairly high. In glioma,the positive ratio rose as the grade rises from 2 to 4 one by one (i.e.,malignancy increases). Further, the DiAcSpm levels per se became fairlyhigh at that time.

EXAMPLE 9

Detection of Primary Malignant Lymphoma of Brain

In this Example, DiAcSpm was detected from samples collected frompatients with primary malignant lymphoma of the brain.

(1) Case 1 (female, age 56, primary malignant lymphoma of the brain)

This patient has undergone cranial irradiation and methotrexate (MTX)large dose treatment against primary malignant lymphoma of the brain asearly treatment. At the start of DiAcSpm measurement, incipient lesionswere in the state of complete remission (CR). However, metastases to thebone marrow and the spleen became evident on images and as conditions attime points (1) and (2) indicated in FIG. 16. At those points, a sharpelevation of DiAcSpm was recognized. This degree of elevation was higherthan that of β-2 microglobulin, a commonly used marker. Since time point(2), reactions to the treatmen (Table 19) began to be seen and theDiAcSpm level was lowered. At time points (3) and (4), treatment againstrecurrence became difficult. Besides, periods of partial remission (PR)became gradually shorter, and the DiAcSpm levels were not completelylowered. At time point (5), tumor was completely showing resistance totreatment, and the DiAcSpm level rose sharply.

Relations between recurrence, treatments and reactions (exitus) areshown in Table 19. In Table 19, items (1) to (5) and a) to f) correspondto those shown in FIG. 16. TABLE 19 Recurrence site Treatment Reaction(1) Spinal dissemination a) Anterior spinal irradiation CR at 36 Gy (2)Metastasis to the spleen b) ESHAP chemotherapy × 3 CR (3) Intracerebralrecurrence c) Fractionated partial irradiation PR (left frontal lobe) at20 Gy (4) Dissemination in the d) Administration of MTX & PR medullarycavity Ara-C into the medullary cavity (5) Intracerebral recurrence e)Fractionated partial irradiation PD (callosum) at 20 Gy f) Ara-C largedose treatment(2) Case 2 (male, age 60, primary malignant lymphoma of the brain)

DiAcSpm levels were high until the start of treatment. Then, tumortissues in the right frontal lobe were removed surgically, followed bycranial irradiation at 40 Gy and 5 cycles of MTX large dose treatment.As a result, complete remission was achieved. At this point, DiAcSpmlevel decreased below the cut-off value.

Therefore, it was shown that DiAcSpm can be a definite marker for thetreatment effect of early treatment of brain tumor.

(3) CONCLUSION

In the diagnosis of malignant lymphoma, problems such as described beloware enumerated.

(i) Even if the tumor can be judged CR on images, there is a highpossibility that tumor cells capable of proliferation are stillremaining in the central nerve;

(ii) Even if the remnant of lesions may be grasped on images, tumorcells may no longer exist; and

(iii) The tumor may metastasize to organs other than the brain.

The present invention can solve above problems. By measuring thepositive ratio of DiAcSpm, it is possible to detect the presence orabsence of tumor cells regardless of lesions grasped on images.Therefore, it becomes possible to judge when and to what extentchemotherapy should be carried out against malignant lymphoma. Besides,by using DiAcSpm as an indicator, it is possible to grasp those caseswhere conversion to malignancy occurs during the process of treatment ofcancer. Therefore, it is possible to diagnose the recurrence of canceror predict prognosis, which enables establishment of appropriatetreatment policy. Further, the kit of the present invention is not onlycapable of screening cancer or making preoperative diagnosis but alsocapable of detecting therapeutic effect, recurrence, conversion tomalignancy, etc. even in those cases where histological diagnosis hasbeen established.

EXAMPLE 10

Detection of Astrocytoma

In this Example, DiAcSpm was measured in a patient (age 36, male) withgrade 3 astrocytoma (a kind of glioma) after he received a surgicaloperation for recurred tumor (FIG. 18).

After the operation, tumor was remaining but urinary DiAcSpm did notshow high levels.

No big changes were observed after irradiation treatment andchemotherapy. Even when radiation necrosis occurred, no elevation ofDiAcSpm was recognized. On the other hand, when the callosal expandedbecause of recurrence and clinical malignancy of tumor was clearlyaccelerated, DiAcSpm level began to rise sharply. In glioma, conversionfrom grade 3 to grade 4 frequently occurs. It is often difficult toprove this conversion by diagnostic imaging or with pathological tissues(e.g., to discriminate between radiation necrosis and recurred lesionsby diagnostic imaging). In the present invention, it was possible todiscriminate between the above-mentioned radiation necrosis and thepresence/absence of recurred lesions by detecting urinary DiAcSpm.Therefore, it was demonstrated that detection of DiAcSpm is extremelyuseful clinically as an indicator of the degree of malignancy.

INDUSTRIAL APPLICABILITY

According to the present invention, DiAcSpm as a tumor marker isprovided. Further, according to the present invention, an antibody inwhich total interference on the measurement results caused by itscross-reaction with DiAcSpm analogues is extremely small and whichspecifically binds to a trace amount of DiAcSpm; and a kit comprisingthe antibody are also provided. The kit of the present invention isextremely useful because this kit is capable of detecting the presenceor absence of various cancers, the degree of malignancy of cancers, thepresence or absence of recurrence, etc. by using the positive ratio ofDiAcSpm as an indicator.

1. A tumor marker comprising N¹,N¹²-diacetylspermine.
 2. A method of detecting a tumor, comprising reacting an antibody to N¹,N¹²-diacetylspermine with a biological sample.
 3. A method of evaluating the state of a tumor, comprising reacting an antibody to N¹,N¹²-diacetylspermine with a biological sample to thereby detect N¹,N ¹²-diacetylspermine, and evaluating the state of the tumor using the resultant detection results as an indicator.
 4. The method according to claim 2, wherein the biological sample is urine.
 5. The method according to claim 2, wherein the tumor is at least one selected from the group consisting of urogenital malignant tumors, colorectal cancer, breast cancer, pancreatic cancer, biliary tract cancer, lung cancer, liver cancer, uterine cervix cancer, brain tumor, myelogenous leukemia and malignant lymphoma.
 6. The method according to claim 3, wherein the state of the tumor is at least one selected from the group consisting of the presence/absence of cancer, the degree of progression of cancer, the degree of malignancy of cancer, the presence/absence of metastasis of cancer and the presence/absence of recurrence of cancer.
 7. The method according to claim 6, wherein the cancer is early cancer.
 8. An antibody to DiAcSpm, which has at least one property selected from the following (a) and (b): (a) cross-reactivity with N¹-AcSpd: 0.1% or less (b) total cross-reactivity with DiAcSpm analogues present in urine: 5% or less.
 9. The antibody according to claim 8, which is a polyclonal antibody or monoclonal antibody.
 10. A tumor detection kit comprising an antibody to N¹,N¹²-diacetylspermine.
 11. The kit according to claim 10, wherein the antibody has at least one property selected from the following (a) and (b): (a) cross-reactivity with N¹-AcSpd:
 0. 1% or less (b) total interference on the measurement results caused by its cross-reaction with DiAcSpm analogues present in urine: 5% or less.
 12. The kit according to claim 10, wherein the antibody is a polyclonal antibody or monoclonal antibody.
 13. The kit according to claim 10, wherein the tumor is selected from the group consisting of urinary tract malignant tumors, large bowel cancer, breast cancer, pancreatic cancer, biliary tract cancer, lung cancer, liver cancer, uterine cervix cancer, brain tumor, myelogenous leukemia and malignant lymphoma.
 14. The kit according to claim 10, which has at least one property selected from the following (a) to (c): (a) lower detection limit in actual measurement: 9.06 nM (b) within-run reproducibility: CV=10% or less (c) between-day reproducibility: CV=10% or less.
 15. The method according to claim 3, wherein the biological sample is urine.
 16. The method according to claim 3, wherein the tumor is at least one selected from the group consisting of urogenital malignant tumors, colorectal cancer, breast cancer, pancreatic cancer, biliary tract cancer, lung cancer, liver cancer, uterine cervix cancer, brain tumor, myelogenous leukemia and malignant lymphoma. 